Test apparatus and methods for ST2 cardiac biomarker

ABSTRACT

The technology described in this document can be embodied in a test strip for use in measuring a level of an ST2 cardiac biomarker in a blood plasma sample. The test strip includes a base, and a plurality of conjugates, wherein each conjugate includes a reporter group bound to a first antibody that binds to ST2. A conjugate pad disposed along a length of the base and is configured to hold the plurality of conjugates that bind with ST2 to produce conjugate-ST2 complexes. The conjugate pad is further configured to receive the blood plasma sample. The test strip also includes a plurality of second and third antibodies that bind to ST2, and the conjugate-ST2 complexes, respectively. The plurality of second antibodies are bound to a membrane in a test location and the plurality of third antibodies are bound to the membrane in a control location.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.16/127,456, filed on Sep. 11, 2018, which is a divisional application ofU.S. patent application Ser. No. 14/566,955, filed on Dec. 11, 2014, nowU.S. Pat. No. 10,079,073, the entire contents of which are herebyincorporated by reference.

TECHNICAL FIELD

The invention relates to detecting the presence of cardiac biomarkers inblood.

BACKGROUND

Biomarkers that indicate a subject's likelihood of being afflicted bycorresponding health-related condition significantly enhance aphysician's ability to make informed treatment decisions.

Tominaga, FEBS Lett. 258:301-304 (1989), describes the isolation ofmurine genes that were specifically expressed by growth stimulation inBALB/c-3T3 cells; they termed one of these genes “St2” (for GrowthStimulation-Expressed Gene 2). The St2 gene encodes two proteinproducts: ST2, which is a soluble secreted form; and ST2L, atransmembrane receptor form that is very similar to the interleukin-1receptors. The HUGO Nomenclature Committee designated the human homolog,the cloning of which was described in Tominaga et al., Biochim. Biophys.Acta. 1171:215-218 (1992), as Interleukin 1 Receptor-Like 1 (IL1RL1).The two terms are used interchangeably herein.

The ST2 gene is a member of the interleukin-1 receptor family, whoseprotein product exists both as a trans-membrane form, as well as asoluble receptor that is detectable in serum (Kieser et al., FEBS Lett.372(2-3):189-93 (1995); Kumar et al., J. Biol. Chem. 270(46):27905-13(1995); Yanagisawa et al., FEBS Lett. 302(1):51-3 (1992); Kuroiwa etal., Hybridoma 19(2):151-9 (2000)). ST2 was described to be markedlyup-regulated in an experimental model of heart failure (Weinberg et al.,Circulation 106(23):2961-6 (2002)) and this observation was validated inmore recent work published by Pascual-Figal, et al. (2014). Analysis ofclinical study cohorts shows that ST2 concentrations may be elevated inthose with chronic severe heart failure (HF) (Weinberg et al.,Circulation 107(5):721-6 (2003)) as well as in those with acutemyocardial infarction (MI) (Shimpo et al., Circulation 109(18):2186-90(2004)) and that this elevated concentration is clinically meaningful.

The trans-membrane form of ST2 is thought to play a role in modulatingresponses of T helper type 2 cells (Lohning et al., Proc. Natl. Acad.Sci. USA, 95(12):6930-5 (1998); Schmitz et al., Immunity 23(5):479-90(2005)), and may play a role in the development of tolerance in statesof severe or chronic inflammation (Brint et al., Nat. Immunol.5(4):373-9 (2004)), while the soluble form of ST2 is up-regulated ingrowth stimulated fibroblasts (Yanagisawa et al., 1992, supra).Experimental data suggest that the ST2 gene is markedly up-regulated instates of myocyte stretch (Weinberg et al., 2002, supra) in a manneranalogous to the induction of the BNP gene (Bruneau et al., Cardiovasc.Res. 28(10):1519-25 (1994)), and has been shown to be involved in thecardiac remodeling process (Sanada, 2007; Seki, 2009)

SUMMARY

This disclosure describes test kits and apparatus for detecting whethera level of ST2 present in a human subject exceeds a threshold condition.The test strips include multiple antibodies that interact with oneanother or with the ST2 if present in a sample, e.g., a blood sample, toprovide an indication of the level of ST2 in the sample. The flow of thesample within the test strips is assisted by a suitably formulatedbuffer. The test strips are disposed within specially designed housingsto form cassettes, the interior of which is configured to createconsistent support of the strip that aids the flow of the sample and thebuffer along the test strip.

In one aspect, this document features test strips for use in measuring alevel of an ST2 cardiac biomarker in a blood plasma sample. The teststrips include a base, and a plurality of conjugates, wherein eachconjugate includes a reporter group bound to a first antibody that bindsto ST2. Each test strip further includes a conjugate pad disposed alonga length of the base and configured to hold the plurality of conjugatesthat bind with ST2 to produce conjugate-ST2 complexes, wherein theconjugate pad is further configured to receive the blood plasma sample.The test strip also includes a plurality of second antibodies that bindto ST2, and a plurality of third antibodies that bind to theconjugate-ST2 complexes. A membrane is disposed on the base such thatthe membrane is in fluid communication with the conjugate pad. Theplurality of second antibodies are bound to the membrane in a testlocation and the plurality of third antibodies are bound to the membranein a control location arranged further from the conjugate pad than thetest location.

In another aspect, this document features test devices that include atest strip having a first end and an opposite second end, and a housingfor the test strip and a radio frequency identification (RFID) tagconfigured to store information associated with the test strip, a firstsection, and a second section. The first section includes an outer faceand an inner face, wherein the inner face of the first section comprisesa channel to receive the test strip along the length of the firstsection. The second section includes an outer face and an inner face,wherein the second section is configured to be attached to the firstsection such that in an attached configuration the inner face of thefirst section faces the inner face of the second section, and the firstand second sections together enclose the RFID tag and the test stripwithin the housing. The second section includes a buffer port configuredto allow a buffer solution to be dispensed to a portion of the teststrip proximate to a first end of the test strip, a test windowconfigured to facilitate imaging of one or both of a test location and acontrol location on the test strip, and a sample port disposed betweenthe buffer port and the test window. The sample port is configured toenable a sample of blood plasma to be dispensed to the test strip. Thesecond section also includes a set of projections disposed on the innerface of the second section between the test window and the sample portsuch that in the attached configuration, wherein each projection in theset of projections is in contact with the test strip. A height of atleast one projection in the set of projections is different from aheight of another projection in the set of projections, and heights ofthe different projections are configured such that, in the attachedconfiguration, the set of projections produces a pressure gradient thatallows a fluid to flow at a predetermined flow rate along the length ofthe test strip between the first and last projections in the set ofprojections.

In another aspect, this document features test systems for ST2 cardiacbiomarker. The test systems include a test strip, a housing for the teststrip, an identification tag, and a reader device. The test strip isconfigured to facilitate detection of a threshold level of ST2 in bloodplasma. The housing includes a first section that includes an outer faceand an inner face, wherein the inner face of the first section includesa channel to receive the test strip along the length of the firstsection. The second section includes an outer face and an inner face,wherein the second section is configured to be attached to the firstsection such that in an attached configuration the inner face of thefirst section faces the inner face of the second section. The first andsecond sections together enclose the test strip within the housing. Thesecond section includes a buffer port configured to allow a buffersolution to be dispensed to a portion of the test strip proximate to afirst end of the test strip, a test window configured to facilitateimaging of one or both of a test location and a control location on thetest strip, and a sample port disposed between the buffer port and thetest window. The sample port configured to enable a sample of bloodplasma to be dispensed to the test strip. The second section alsoincludes a set of projections disposed on the inner face of the secondsection between the test window and the sample port such that in theattached configuration, each projection in the set of projections is incontact with the test strip. A height of at least one projection in theset of projections is different from a height of another projection inthe set of projections, and heights of the different projections areconfigured such that, in the attached configuration, the set ofprojections produces a pressure gradient that allows a fluid to flow ata predetermined flow rate along the length of the test strip between thefirst and last projections in the set of projections. The reader deviceis configured to accept at least a portion of the housing within thereader. The identification tag is disposed on or within the housing,wherein the identification tag is configured to store informationassociated with the test strip. The reader device includes an opticalsystem that images one or both of the test location and the controllocation on the test strip, and displays an estimated level of ST2 inthe sample of blood plasma.

Implementations of the above aspects can include one or more of thefollowing.

Either or both of the first or the second antibodies can bindspecifically to ST2. The test strip can include an absorbent paddisposed on the base, and in fluid communication with the membrane at anend or side of the membrane opposite the conjugate pad. The absorbentpad can be configured to absorb plasma and buffer that has traversedthrough the membrane. The conjugate pad can be disposed on the base toreceive a buffer solution. The conjugate pad can include glass and/orpolyester fibers. At least one of the plasma separation pad or themembrane can include nitrocellulose. The first antibodies can includemonoclonal antibodies. The reporter group can include fluorescentparticles. The monoclonal antibodies can include 7E4-monoclonal-anti-ST2antibodies that are conjugated to fluorescent particles. The secondantibodies can include 9F8-monoclonal-anti-ST2 antibodies. The thirdantibodies can include Goat anti-Mouse IgG antibodies. The secondantibodies may change in appearance depending on an amount of the boundconjugate present in the blood plasma traversing the first portion.

The predetermined flow rate can be such that the fluid flows from theportion of the test strip adjacent to the buffer port of the secondsection to the portion of the test strip adjacent to the test window ofthe second section in about 20 minutes. The set of projections can be aset of ridges. The distances between ridges can be substantially equal.The set of projections can include four ridges. A first ridge can bedisposed closer to the sample port than the other ridges, a fourth ridgecan be disposed closer to the test window than the other ridges, and asecond ridge and a third ridge can be disposed between the first andthird ridges. The first section can include multiple attachmentprojections that are configured to attach to corresponding attachmentreceptacles disposed on the second section, wherein dimensions of theattachment projections and attachment receptacles are configured suchthat in the attached configuration, the set of projections produces thepressure gradient that allows the fluid to flow at the predeterminedflow rate along the length of the test strip. The reader device can beconfigured to query the identification tag to obtain at least a portionof the stored information.

The technologies described herein provide a number of advantages. Forexample, the new methods and devices can be used to determine whether apatient should be admitted or held as an inpatient for furtherassessment, regardless of whether a definitive diagnosis has been made.Risk stratification based on ST2 level of a given subject can befacilitated, e.g., to make decisions regarding the level ofaggressiveness of treatment that is appropriate for the subject. Bettertreatment decisions can be made, which in turn can lead to reducedmorbidity and mortality, and better allocation of health care resources.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a top view of an example of an ST2 detection apparatus.

FIG. 1B is a perspective view of an example of an ST2 detectionapparatus.

FIG. 2A is a top perspective view of one example of a top portion of theST2 detection apparatus of FIG. 1 .

FIG. 2B is a view of the inner face of the top portion shown in FIG. 2A.

FIG. 2C is a side view of an example of a top portion of the ST2detection apparatus.

FIG. 2D is a view of the inside face of the top portion of FIG. 2C.

FIG. 2E is a cross sectional view of an example of a top portion,together with a blown-up view of the projections.

FIG. 3A is a perspective view of the inner face of an example of abottom portion of the ST2 detection apparatus of claim 1.

FIG. 3B is a top view of the inner face of the bottom portion shown inFIG. 3A.

FIG. 3C is a side view of the inner face of the bottom portion shown inFIG. 3A.

FIG. 4A is a schematic that shows an example of an ST2 test strippositioned in the bottom portion of the ST2 detection apparatus of FIG.1 .

FIG. 4B is a schematic diagram showing construction details of oneexample of a test strip.

FIG. 4C is a schematic diagram showing construction details of anotherexample of a test strip.

FIG. 5A is a photo that illustrates the application of a blood sample tothe apparatus of FIG. 1 .

FIG. 5B is a photo that illustrates the application of a buffer solutionto the apparatus of FIG. 1 .

FIG. 6A is a photo that shows an example of a test result in which acontrol line can be seen, but no test line is visible.

FIG. 6B is a photo that shows an example of a test result that shows acontrol line and a faint test line.

FIG. 6C is a photo that shows an example of a test result that shows aclearly visible control line and a clearly visible test line.

FIG. 7A is a view of the front of a reader device used in analyzing testresults using ST2 test strips.

FIG. 7B is a view of the back of a reader device of FIG. 7A.

FIG. 7C is a top view of the reader device of FIG. 7A in operation.

DETAILED DESCRIPTION

Clinical evaluation of patients, particularly patients with non-specificsymptoms such as dyspnea or chest pain, is often challenging. Thecardiac biomarker ST2 can be used in prognostic evaluation of patients,regardless of the underlying cause of their disease. In some cases, thelevel of ST2 in blood can be a powerful indicator of cardiac health, andsuch information may be used in taking measures to prevent the onset ofacute conditions or even death. The ST2 test apparatus described hereinallows fast and reliable detection of ST2 levels in blood, which canthen be used by physicians and clinicians to determine the besttreatment plan for the patient.

The mRNA sequence of the shorter, soluble isoform of human ST2 can befound at GenBank Acc. No. NM_003856.2, and the polypeptide sequence isat GenBank Acc. No. NP_003847.2; the mRNA sequence for the longer formof human ST2 is at GenBank Acc. No. NM_016232.4; the polypeptidesequence is at GenBank Acc. No. NP_057316.3. Additional information isavailable in the public databases at GeneID: 9173, MIM ID #601203, andUniGene No. Hs.66. In general, the methods, devices, and systemsdescribed herein measure the soluble form of the ST2 polypeptide.

FIGS. 1A and 1B show top and perspective views of an example of an ST2detection apparatus 100. In some implementations, the apparatus 100includes a top portion 105 that is attached to a bottom portion 110. Thetop portion 105 and the bottom portion 110 together form a housing (alsoreferred to as a cassette) within which an ST2 detection test strip isdisposed. In some implementations, the top portion 105 includes a testwindow 115. The test window 115 is an opening or hole in the top portion105 that exposes a portion of the test strip disposed in the housing.The portion of the test strip exposed by the test window 115 includesone or more marker locations that change appearance during the ST2detection test. For example, the marker locations can include a controlline (a location of which can be marked by the letter “C”) thatindicates that a test has been properly conducted, and a test line (alocation of which can be marked by the letter “T”) that becomes visibleif a particular biomarker (e.g., ST2) is present in the analyte. Theshape and dimensions of the test window 115 can be configured such thateach of the marker locations is visible through the test window 115.

The top portion 105 also includes a sample port 120 through which theanalyte (e.g., blood or other body fluid) is dispensed into theapparatus 100. The sample port 105 is an opening or hole in the topportion 105 that allows the analyte to be dispensed on sample-receivingportion of the test strip disposed in the housing. The shape anddimensions of the sample port are configured in accordance with thesample-receiving portion of the test strip. In some implementations, thesample port 120 includes a sidewall 122 that can form a seal with thesample-receiving portion of the test strip. In such a sealingconfiguration, the sidewall 122 inhibits lateral flow of a sampledispensed into the sample port 120 along the top of the test strip.

The top portion also includes a buffer port 125 through which a buffersolution can be dispensed into the apparatus 100. The buffer solutionflows through the test strip, e.g., by capillary action, from thelocation beneath the buffer port 125 in the direction of the test window115. As the buffer solution flows along within the test strip, thesolution provides mobility to the plasma from the blood sample such thatthe plasma also flows along within the test strip from the locationbeneath the sample port 120 towards the test window 115. As the plasmaflows past the portion of the test strip exposed by the test window 115,one or more of the marker locations (e.g., the test line and the controlline) may change in their visual appearance depending on the level ofST2, if any, in the plasma. For example, if the level of ST2 in theplasma is above a threshold level, the marker locations corresponding toboth the test line and the control line change appearance and both linesbecome visible. On the other hand, if the level of ST2 in the plasma isbelow the threshold level, only the marker location corresponding to thecontrol line changes appearance and hence the control line (and not thetest line) becomes visible. The lack of a control line can indicate thatthe plasma has not flowed through the test strip all the way to thecontrol line, and the test is invalid.

In some implementations, in lieu of (or in addition to) a visualdetermination, the level of ST2 in the plasma can be measuredquantitatively. In such cases, the housing or cassette can be configuredto be inserted into a reader device (e.g., the reader device describedbelow with reference to FIGS. 7A and 7B) that analyzes the test stripand provides a quantitative measure of the level of ST2 in the plasma.In some implementations, the reader analyzes the test strip through thetest window 115 (for example, by obtaining an image of the portion ofthe test strip exposed at the test window 115). In some implementations,cassettes that are inserted into a reader may include an opening throughwhich the test strip can be extricated from the cassette by the readerfor analysis.

In some implementations, the level of ST2 can be performed, for example,by analyzing an image of the test line and the control line. In someimplementations, such image analysis can be performed, for example,using an application installed on a computing device such as a laptop ordesktop computer or a mobile device such as a smart phone or tablet. Insome implementations, a user may be able to capture an image of the testline and control line using, for example, a camera of a mobile device.The captured image can then be analyzed, for example, using anapplication installed on the mobile device. In some cases, the capturedimage can also be analyzed by providing the image to a remote computingdevice that executes a suitable image analysis application.

In some implementations, the top portion 105 includes a designatedportion for marking the cassette with identification information relatedto the corresponding subject or patient. In some implementations, thehousing or cassette can also include an automatic identification modulesuch as a radio frequency identification (RFID) tag encoded with theidentification information related to the corresponding sample orpatient. In such cases, the reader includes a suitable module forquerying and retrieving information from the automatic identificationmodule. For example, if the cassette includes an RFID tag, the receivercan be configured to include an RFID reader to retrieve information fromthe tag. Other suitable communication technologies such as near-fieldcommunications (NFC) or Bluetooth® can also be used in place of RFID.

FIGS. 2A-2E show construction details of a particular implementation ofthe top portion 105. FIG. 2A shows the outer face 130 of the top portion105, and FIG. 2B shows the inner face 135 of the top portion 105. Insome implementations, the inner face 135 (that faces the inner face ofthe bottom portion 110) can include multiple mating projections 150 thatare configured to couple with corresponding receptacles in the bottomportion 110. The example of FIG. 2B shows six such mating projections150. Other implementations may have a different number of such matingprojections 150. The mating projections couple with the correspondingreceptacles in the bottom portion 110 such that the top and bottomportions together form a substantially sealed housing or cassette inwhich the test strip is disposed. For this reason, the matingprojections 150 can be referred to as attachment projections, and thecorresponding receptacles can be referred to as attachment receptacles.

In some implementations, the inner face 135 of the top portion 105includes multiple projections 152 a, 152 b, and 152 c (152, in general).In some implementations, the projections can be in the form of ridges.The dimensions of the projections 152 are different from one another,and are configured in accordance with the varying thickness of the teststrip housed within the cassette. In some implementations, the teststrip is thicker in the portion that comes in contact with projection152 a than the portions that come in contact with portions 152 b and 152c, respectively. Accordingly, the height of the projections 152 from theinner face 135 can be adjusted such that projection 152 a is shorterthan projection 152 b, and projection 152 b is shorter than projection152 c. The respective height of each projection 152 is configured suchthat when the top portion 105 is coupled with the bottom portion 110 toform a cassette, each of the projections 152 is in contact with the teststrip housed within the cassette. Further, the set of projections 152are configured such that they produce a pressure gradient within thetest strip to allow a sample fluid to flow at a predetermined flow ratealong the length of the test strip between the projection 152 a and theprojection 152 c. In some implementations, the set of projections 152can be configured to support the test strip without creating a pressurepoint that hinders the flow rate along the length of the test strip. Insome implementations, the projections can be measured with respect to abaseline such as the line 153. In the example of FIG. 2E, the heights ofthe projections 152 a, 152 b, and 152 c are 0.003 units, 0.011 units,and 0.027 units, respectively. The flow rate can also be configured, forexample, by varying various parameters of the test strip, including, forexample, composition of conjugates disposed in the test strip.

Other variations of the top portion are also possible. FIGS. 2C and 2Dshow the side view and the inside face 136, respectively, of an exampleof such a variation 106. The top portion 106 shown in FIGS. 2C and 2D,can be used, for example, in an implementation where the cassette isused in conjunction with a reader device such as the one described belowwith reference to FIGS. 7A-7C. In some implementations, where thecassette is used in conjunction with a reader device, plasma (and notwhole blood) can be used as the test specimen or sample, and the flowdynamics can be different from implementations that use whole blood asthe sample. Accordingly, the sample port 121 of the top portion 106(FIG. 2D) can be made smaller than the sample port 120 of the topportion 105 (FIG. 2B), where a larger port may be needed to facilitatean appropriate flow for separating plasma from the whole blood sample.

In some implementations, the top portion 105 and the bottom portion 110also enclose an identification tag (such as a radio frequencyidentification (RFID) tag) that includes identification informationabout a corresponding patient and/or sample. In such cases the topportion 105 may have a particular portion 151 configured for receivingthe identification tag. In some implementations, the identificationinformation can also be encoded, for example, as a barcode or quickrecognition (QR) code, and printed on an exterior face of the topportion or the bottom portion. The identification tag or code can bescanned or detected by an appropriate reader to automatically determinedidentification information related to a patient or sample.

FIG. 2E shows a cross sectional view of the top portion 105, togetherwith a blown-up view of the projections 152. As shown in FIG. 2E, theheight of the projection 152 b is more than that of the projections 152a, and the height of the projection 152 c is more than that of theprojections 152 b. In the example shown in FIG. 2E, to achieve thedesired pressure gradient within a test strip in the cassette, aproportion of the heights of the projections 152 a, 152 b, and 152 cfrom a reference level 155 is 3:11:27. In some implementations, the teststrip disposed within the cassette may have a limited range of pressuretolerance against leakage. For example, if the fit is too tight,portions of the test strip may be crushed thereby resulting in leakageof test fluid from the strip. On the other hand, if the fit is tooloose, there may be leakage too. In some implementations, the heights ofthe projections 152 a, 152 b, and 152 c can be configured such that thetest strip is held within the cassette without breaching thecorresponding pressure tolerance range. This can prevent leakage fromthe test strip disposed within the cassette.

FIGS. 3A-3C show construction details of the bottom portion 110 of theapparatus 100. FIG. 3A shows a perspective view in which the inner face160 of the bottom portion is visible. When the bottom portion 110 isattached to the top portion 105, the inner face 160 faces the inner face135 of the top portion 105. As shown in FIG. 3C, the outer face 165 ison the opposite surface of the bottom portion 110, and is not visible inthe view shown in FIG. 3A. The inner face 160 includes multiplereceptacles 175 that are configured to couple with the matingprojections 150 disposed on the inner face 135 of the top portion toform the cassette that houses the test strip. In some implementations,the receptacles 175 are circular, and the inner diameters of thereceptacles 175 are marginally smaller than the diameters of thecorresponding mating projections 150. In the example shown, the innerdiameter of the receptacles are 0.047 units, whereas the diameter of themating projections are 0.05 units (see FIG. 2B). This allows for a tightcoupling between the mating projections and the correspondingreceptacles.

As shown in FIG. 3B, the inner face 160 includes a first channel 177 anda second channel 178 that together support the test strip within thecassette. In some implementations, the first channel 177 includes araised portion 179 that has a groove 180 that forms a sealingconfiguration with the buffer port 125 of the top portion 105. In someimplementations, the inner face 160 of the bottom portion 110 can alsobe configured to include one or more supporting portions for supportingthe test strip. For example, the inner face 160 of the bottom portion110 can include one or more supporting platforms 182. In someimplementations, the first channel 177 and/or the second channel 178 canbe configured to include one or more supporting projections (e.g., thesupporting projection 185 in the first channel 177) to support the teststrip. The bottom portion 110 can also include a grip 186 for holdingthe cassette. In some implementations, the grip 186 can be corrugated toreduce the chance that the cassette will slip from a person's hand.

FIG. 4A shows an example of a ST2 test strip positioned in the bottomportion 110 of the apparatus 100. In the example shown in FIG. 4A, thetest strip is supported by the first channel 177, the second channel178, and the supporting structure 182. Various types of test strips canbe used in the apparatus 100. Two examples of such test strips aredescribed below with reference to FIGS. 4B and 4C.

FIG. 4B shows the construction details of an example of a test strip405. The test strip 405 can include a base 407 that provides structuralsupport. For example, the base can be constructed from an 80 mm thicklamina (e.g., made of plastic, e.g., polyvinyl chloride (PVC),polystyrene, polyester, or biodegradable plastic such as celluloid) onwhich the other portions of the test strip are laminated. The test stripincludes a conjugate pad 409 and a membrane 410. The conjugate pad 409and the membrane 410 are disposed on the base 407 such that the membrane410 is in fluid communication with the conjugate pad 409. The conjugatepad 409 can be composed of an absorbent filtration media (e.g., a 38 mmGrade 8964 pad manufactured by Ahlstrom Corporation). In someimplementations, the conjugate pad can include glass and/or polyesterfibers. The conjugate pad 409 includes one or more conjugates 412 thatbind with ST2 present in a body fluid (e.g., plasma) to produceconjugate-ST2 complexes. The conjugates can include, for example, areporter group bound to antibodies that bind to ST2. In someimplementations, the antibodies that bind to ST2 can include monoclonalantibodies such as 7E4 or 9F8-monoclonal-anti-ST2 antibodies.

The reporter group can include, for example, gold particles, and in suchcases the antibodies are conjugated to colloidal gold. The reportergroup can also include fluorescent particles (e.g., fluorophores such asfluorescein, rhodamine, or eosin) for implementations in which afluorescent assay is used. As one particular example, the 7E4 monoclonalanti-ST2 antibody can be conjugated to 40 nM colloidal gold at 0.010mg/ml of 1 OD colloidal gold. As another particular example, the 9F8antibody can be conjugated to gold or fluorescent particles for use in afluorescent assay.

In some implementations, the conjugate pad may be pre-treated to includea conjugate block buffer. The conjugate block buffer includes abuffering agent, such as borate or N-(2-Acetamido)-2-aminoethanesulfonicacid (ACES), Tris-HCL, Tris-base, 3-(N-morpholino)propanesulfonic acid(MOPS), phosphate buffered saline (PBS), and a blocking agent (e.g.,bovine serum albumin (BSA), casein, Fish Gelatin, Polyethylene glycol(PEG), Polyvinyl alcohol (PVA), Polyvinylpyrrolidone (PVP), Polyacrylicacid (PAA), Polyacrylic maleic acid (PAMA) to block non-specific bindingof the antibodies used in the test assay.

In on example, the conjugate block buffer can include, for example, asolution of 50 mM Borate at 10% Bovine Serum Albumin (BSA) and havingpH=9.0. In another example, the conjugate block buffer can include asolution of 100 mM ACES, 25 mM NaCl, 75 mM MgCl₂, at 3% BSA, 1%Polyvinylpyrrolidone (ave. MW 40K) (PVP-40), 0.25% Triton X-100, pH 6.5.In yet another example, the conjugate block buffer can include asolution of 10 mM Borate, 3% BSA, 0.25% PVP-40, 0.25% Triton X-100,pH=8.0. In some implementations, a conjugate diluent of 50 mM Borate at1% BSA, and having a pH=9.0 can also be used.

The conjugate can be pre-treated to include the conjugate block bufferby dipping and soaking the conjugate pad in the conjugate block bufferfor a period of time (e.g., two minutes). The excess buffer can then beremoved from the conjugate pad, for example, by placing the conjugatepad between layers of absorbent material (e.g., paper towels) andapplying pressure. The wet conjugate pads can then be dried (e.g., at37° C. for one hour) and stored desiccated (<20% RH) at roomtemperature.

The conjugates 412 can be added to the conjugate pad 409, for example,by spraying the conjugate pad with a solution including the conjugates.The conjugate pad 409 is then dried (e.g., in a 37° C. forced air ovenfor one hour) and stored desiccated (<20% RH) at room temperature. Theconjugate pad 409 can be trimmed to the appropriate size and laminatedonto the base 407.

The solution including the conjugates that is used for spraying theconjugate pad 409 can be prepared in various ways. In general terms theprocess is as follows. First, the antibody is dialyzed, e.g., with 10 mMphosphate (pH ˜7.3), and an effective amount of the reporter group,e.g., 40 nm colloidal gold, is adjusted to a relatively neutral pH,e.g., from about 5.0 to about 10.0, e.g., 6.5 to 9.5, e.g., the pH canbe 7.0, using a buffering agent, e.g., as noted above, e.g., 0.2 MK2CO3. An effective amount of the antibodies, e.g., 10 μg of theantibody, is then added to an amount of the reporter group mixture,e.g., 1 ml of colloidal gold, and the solution is mixed for a timesufficient to thoroughly mix all the components, e.g., for 15 minutes atroom temperature. Then the blocking agent is added, e.g., 10% BSA in abuffering agent, e.g., 50 mM borate (pH ˜9.0) using 10% of the volume ofthe reporter group, e.g., colloidal gold, used, and the solution ismixed again, e.g., for 30 minutes at room temperature. The solution isthen centrifuged for about 30 minutes at 14,000×G to form a pellet ofthe components that have not dissolved. After the supernatant is removedand discarded, the pellet is re-suspended in a conjugate diluent and anappropriate amount is added to reach a target optical density (OD). Insome cases, a sugar, e.g., sucrose and/or trehalose can be added to thegold conjugate in appropriate amounts (e.g., 20% and 5%, respectively)and mixed until dissolved.

In some implementations, the test strip can also include a sample pad414 on which the sample (e.g., whole blood) is dispensed. The sample pad414 can be disposed over the conjugate pad 409, and can be configured toallow a part of the sample to pass through on to the conjugate pad 409.For example, if the sample used for the test strip is whole blood, thesample pad 414 can be configured to allow blood plasma to pass throughwhile blocking other constituents of the blood. For this reason, thesample pad 414 can also be sometimes referred to as a plasma separationpad. In some implementations, a plasma separation membrane such as theVivid™ plasma separation membrane manufactured by Pall Corporation canbe used as the sample pad 414.

In operation, a sample (e.g., plasma) received within the conjugate pad409 flows from the conjugate pad 409 to the membrane 410 and traversesthe length of the membrane 410. The test strip 405 also includes anabsorbent pad 416 (sometimes also referred to as a wick pad) forcollecting the residual sample coming out of the membrane. In someimplementations, a CO95 pad manufactured by EMD Millipore Corporationcan be used as the absorbent pad 416. In other implementations, a CO83pad, also manufactured by EMD Millipore Corporation, can be used as theabsorbent pad 416. The absorbent pad 416 and the conjugate pad 409 aredisposed at opposite ends (along the length) of the membrane 410.

Various combinations of the constituent parts described above can beused in constructing the test strip. For example, to construct the teststrip 405 shown in FIG. 4B, the membrane 410 is laminated over the base407. A CO83 absorbent pad 416 (e.g., a 21 mm or 25 mm wicking pad) canthen be placed at one end of the backing card with an overlap (e.g., a 2mm overlap) with the membrane 410. The conjugate pad 409 (e.g., a 36 mmor 38 mm conjugate pad) is laminated onto the base 407 overlapping themembrane by a short distance to ensure a good contact, e.g., 2 mm. Thesample pad 414 (e.g., a 26 mm Vivid™ blood separation pad) is thenlaminated on top of the conjugate pad 409. Two strips of cover tape 420can then be placed at both ends of the sample pad 414 such that onestrip of the cover tape overlaps the membrane and the other strip of thecover tape overlaps the conjugate pad. The sheet thus prepared can thenbe cut into strips that fit the channel within the cassette.

In some implementations, the test strip does not include a sample pad.An example of such a test strip 450 is shown in FIG. 4C. To constructthe test strip 450, the process followed can be substantially similar tothe one followed for constructing the test strip 405, with the exceptionthat no sample pad or cover tapes are used. The test strip 450 can alsobe used as dipsticks.

In both test strips 405 and 450, the ST2 detection assay is carried outon the membrane 410. In some implementations, a nitrocellulose membrane(e.g., HF135 manufactured by EMD Millipore Corporation) can be used asthe membrane 410. The membrane 410 includes a test location 422 and acontrol location 424. The test location 422 includes an antibody thatbinds specifically to any ST2 in the sample, such as 7E4 or 9F8monoclonal anti-ST2 antibodies at a predetermined concentration (e.g.,0.75 mg/mL, 1 mg/mL, 1.5 mg/mL, or 2.0 mg/mL). The control locationincludes another antibody that binds specifically to some component ofthe conjugate, such as the anti-ST2 antibody, which can be bound by agoat anti-mouse IgG at a predetermined concentration (e.g., 2.0 mg/mL,0.5 mg/mL, or 0.125 mg/mL). The test location 422 and the controllocation 424 are striped onto the membrane using, e.g., a frontlinedispenser and at a predetermined dispense rate (e.g., 1 μL/cm). Themembrane is then dried and stored desiccated at room temperature.

In some implementations, the membrane 410 is pre-treated using amembrane blocking buffer. An example of a solution used as the membraneblocking buffer is 100 mM Sodium Phosphate, 0.1% Sucrose, 0.1% BSA, and0.2% PVP-40, at pH 7.2. Other blocking agents are known and can be used.Examples of such blocking agents include bovine serum albumin (BSA) andcasein, dry milk, fish skin gelatin, and polyethylene glycol (PEG). Topre-treat the membrane, the membrane 410 can be slowly dipped into themembrane blocking buffer and allowed to wick across the membrane 410.Excess buffer can be blotted off the top of the membrane 410, forexample, using a paper or cloth, e.g., a Kimwipe™. The membrane 410 canthen be dried (e.g., at 37° C. for 30 minutes), removed, and storeddesiccated (<20% RH) at room temperature until used.

To perform a test using the apparatus 100 described above, a test strip(e.g., the test strip 405) can be placed within the apparatus 100 and apredetermined amount of test fluid (e.g., blood or plasma) is dispensedinto the sample port 120. This is shown in FIG. 5A. In general,commercial cassettes will be preloaded with the appropriate test strip.For example, 40 μl of blood or 30 μl of plasma can be dispensed into thesample port 120 using a pipette, as shown in FIG. 5A. In someimplementations, the sample may be pre-treated with a human anti-mouseantibody (HAMA) blocker. After waiting for a predetermined time period(e.g., about 1 minute) for the sample to soak in, a running buffer isdispensed into the buffer port 125. This is shown in FIG. 5B. For 40 μlof blood or 30 μl of plasma, about 120 μl of the running buffer may bedispensed into the buffer port 125. In some implementations, theconjugate 412 (as shown in FIG. 4B) is not already present within theconjugate pad 409, and is also dispensed into the buffer port 125. Ifthe conjugate 412 is already present within the conjugate pad 409, onlythe running buffer is dispensed into the buffer port 125. Within theconjugate pad 409, the ST2 in the sample binds with the conjugate 412 toproduce conjugate-ST2 complexes. These conjugate-ST2 complexes traversethe conjugate pad 409 and the membrane 410 and get bound to antibodiesin the control location 424. If the level of ST2 in the sample is abovea threshold, not all of the ST2 is bound to the conjugate 412. Theunbound ST2 traverses the conjugate pad 409 and the membrane 410 wherethey get bound to the antibodies at the test location 422.

The strip is read or otherwise evaluated after another predeterminedtime period (e.g., about 5 to 25 minutes, e.g., 10 to 20 minutes, e.g.,15 minutes). If a fluorescent assay is used, the test strip can beevaluated using a fluorescence reader, e.g., an ESE Fluorescent Reader.If a gold assay is used, the test results are visually inspected andsubjectively graded using, for example, a scale calibrated to thespecific test.

The running buffer dispensed into the buffer port 125 is formulated tofacilitate and/or expedite the flow of the sample (e.g., plasma) throughthe conjugate pad 409 and the membrane 410. The running buffer generallyincludes a buffering agent such as N-(2-Acetamido)-2-aminoethanesulfonicacid (ACES), together with other components (e.g., detergents such asTween-20 and Triton X-100). One example composition for the runningbuffer can be 100 mM ACES, a salt solution that can be used to achieve adesirable ionic composition of the buffer (e.g., 100 mM MagnesiumChloride), 0.1% Tween-20, 0.05% Proclin 300, with pH about 6.5.

FIGS. 6A-6C show examples of test results that are visually inspected todetermine the presence or absence of ST2 in the sample. FIG. 6A shows aresult in which the control line 605 can be seen (therefore indicatingthat the test was successfully completed), but no test line is visible.Such absence of a visible test line may indicate that the level of ST2is below the cutoff level (e.g., 35 ng/mL) that the apparatus isconfigured to detect. FIG. 6B shows the control line 605, and a fainttest line 610. The faint test line 610 can indicate that the level ofST2 in the corresponding sample is close to the cutoff level butsignificantly above the cutoff level. FIG. 6C shows a clearly visiblecontrol line 605 and a clearly visible test line 610. The clear testline 610 indicates that the level of ST2 in the corresponding sample isabove the cutoff level.

In some implementations, a reader device can be used for analyzing testresults for tests performed using the ST2 test strips described above.FIGS. 7A and 7B show the front and back, respectively, of an example ofsuch a reader device 700 developed by LRE Medical GmbH, Germany. Thereader device 700 includes a power switch 725 and a receiving section750 as shown in FIG. 7C. The receiving section 750 can be configured toaccept at least a portion of the cassette or housing within which a teststrip is disposed. For example, the receiving section 750 can include aslide-out section that accepts the cassette. In another example, thereceiving section can include an opening through which a portion of thecassette is inserted into the reader device 700. In someimplementations, the reader device 700 may be configured to initiate ananalysis of the test strip automatically upon insertion of the cassetteinto the reader. The operation can also be controlled using a commandprovided via a user interface.

In some implementations, the reader device 700 can include, for example,an optical system for analyzing the amount of analyte in an ST2 teststrip. For example, the reader device can include a fluorescence opticalsystem configured for a particular operating range such as TF5 (i.e.,excitation at ˜650 nm and emission at ˜670 nm) to quantitativelydetermine the amount of analyte present. The optical system may beoptimized for the particular operating range, and/or the type offluorescence that is to be analyzed. In some implementations, the readerdevice 700 can be made user-configurable, for example via a graphicaluser interface (GUI), to handle multiple different types of testcartridges with different assays.

In some implementations, the reader device 700 can include a display(e.g., a 3.5″ LCD QVGA color graphic display with backlighting) and akeypad 704 for operating the device. The key pad 704 can include, forexample, any combination of soft keys, functional keys (e.g., eject,main menu, paper feed), navigation keys (e.g., up, down, left, right),character keys, and numeric keys. The device 700 can also include one ormore on-board controllers that schedule, manage, and drive variousmotors, actuators, sensors, etc. in order to analyze test strips andprovide results. In some implementations, the GUI can be used forpresenting one or more menu-driven interfaces to support one or morefunctions such as running tests, performing quality control, retrievalof stored results, querying databases, performing system checks,facilitating instrument setup, and facilitating assay development.

In some implementations, the reader device 700 can include anillumination optic system configured to emit electromagnetic radiationthat impinges the portion of the test strip being evaluated. The readerdevice also includes a receiver optic system configured to detectportions of the electromagnetic energy reflected, refracted, absorbed,emitted, and/or transmitted through the test strip. In someimplementations, the reader device 700 can include a camera module forcapturing image information pertaining to the portion of the test stripbeing evaluated. The reader device 700 can also include one or moreprocessors for analyzing the information obtained by the illuminationoptic system, receiver optic system, and/or the camera module. In someimplementations, the reader device 700 can also include anidentification module (e.g., an RFID tag reader, or barcode reader) toautomatically determine identification information from the cassette orhousing inserted into the housing. The reader 700 can also include oneor more of an acoustic output device (e.g., a speaker), an internalprinter (e.g., for printing results), a temperature sensor, a datastorage device (e.g., random access memory, hard disk etc.), and one ormore communication ports. Examples of communication ports 730 are shownin FIG. 7B, and can include USB host interfaces (USBH), local areanetwork (LAN) interface, PS2 interface, and USB device interface (USBD).The reader device 700 can also be configured to have wirelesscommunications capabilities.

The information captured by the optic systems within the reader device700 can be analyzed to obtain quantitative information on analyteswithin a test strip. For example, the information can be analyzed todetermine a level of ST2 within a sample tested using the test strip.The analysis can be performed, for example, on board the reader device700 using one or more processors of the device, or at a remote computingdevice with which the reader device 700 communicates.

The analysis can include various processes. In some implementations, thecaptured information can be analyzed to determine a level of darknessfor the test line and/or the control line of the test strip, and thelevel of darkness is correlated to an amount of ST2 in the sample. Insome implementations, the level of ST2 is determined using multiple teststrips for analyzing the sample from the same individual.

In some implementations, for each run, the test strip is read multipletimes, possibly at substantially periodic intervals. For example, foreach run of a test, a corresponding test strip can be read once everyminute ten times to obtain ten readings. Absolute differences can thenbe calculated between each pair of readings, and the results stored asan appropriate data structure such as a matrix. For the example of tenreadings, the size of the matrix would be 10×10. The rows and columns ofthe matrix are then summed and the results sorted. The values thatcorrespond to the smallest differences can then be selected forcomputing the representative level of darkness for the test strip. Forexample, five values corresponding to the smallest differences can bechosen and then averaged to determine the representative level ofdarkness used in determining the amount of ST2 in the correspondingsample.

Using the apparatus and test strips described herein, ST2 levels in thebody can be easily and reliably determined. Such determination isbeneficial at least because elevated concentrations of ST2 are markedlyprognostic for death within one year, with a dramatic divergence insurvival curves for those with elevated ST2 soon after presentation,regardless of the underlying diagnosis. As one example, there is adramatic relationship between elevations of ST2 and the risk formortality within one year following presentation with dyspnea. Therelationship between ST2 and death in dyspneic patients can beindependent of diagnosis, and supersede all other biomarker predictorsof mortality, including other markers of inflammation, myonecrosis,renal dysfunction, and notably NT-proBNP, a marker recently described ashaving value for predicting death in this population (Januzzi et al.,Arch. Intern. Med. 2006; 166(3):315-20). Indeed, most of the mortalityin the study was concentrated among subjects with elevated ST2 levels atpresentation; however, the combination of an elevated ST2 and NT-proBNPwas associated with the highest rates of death within one year.

Elevated concentrations of ST2 can also be correlated with the presenceof severe disease in a subject, regardless of the underlying cause ofthe disease. Therefore, for undiagnosed subjects, the apparatusdescribed herein can be used to determine how aggressively a diagnosisshould be sought. For example, a high ST2 level would indicate thepresence of severe disease, and suggest that the subject should betreated as a high-risk case. For subjects with a known diagnosis, theapparatus described herein can be used to help determine the severity ofthe underlying pathology because a higher ST2 level is associated withmore severe disease.

The test strip, and the apparatus in general can be used in assessingprognosis and monitoring the efficacy of treatment of variouscardiovascular diseases. The use of ST2 as a marker for diseases hasbeen described in the following U.S. patents and Published Applications,the contents of which are incorporated herein by reference: US2009/0305265 (Snider et al.), US 2010/0009356 (Snider et al.), US2011/0053170 (Snider et al.), U.S. Pat. No. 8,597,958 (Lee), U.S. Pat.No. 8,617,825 (Snider et al.), US 2014/0045200 (Snider et al.), US2012/0065897 (Snider et al.), U.S. Pat. No. 7,432,060 (Lee), U.S. Pat.No. 7,655,415 (Lee), U.S. Pat. No. 7,670,769 (Lee), U.S. Pat. No.7,985,558 (Lee), U.S. Pat. No. 8,420,785 (Snider et al.), US2010/0055683 (Snider et al.), U.S. Pat. No. 8,530,173 (Lee), US2013/0273562 (Lee), U.S. Pat. No. 8,734,769 (Lee), U.S. Pat. No.7,989,210 (Lee), U.S. Pat. No. 7,998,683 (Snider et al.), U.S. Pat. No.8,090,562 (Snider et al.), US 2013/0177931 (Snider et al.), US2013/0244236 (Snider et al.), US 2012/0276551 (Snider), US 2014/0058743(Snider et al.), US 2013/0071404 (Snider et al.), US 2013/0345805(Snider et al.), U.S. application Ser. No. 14/244,526, filed Apr. 3,2014 (Snider et al.), US 2014/0051773 (Snider), US 2012/0040381 (Snideret al.), U.S. application Ser. No. 14/267,487 (Snider), and US2013/0317030 (Lee).

Other Embodiments

The foregoing description is intended to illustrate and not limit thescope of the appended claims. Other aspects, advantages, andmodifications are within the scope of the following claims.

What is claimed is:
 1. A test system comprising: a test strip; a housingfor the test strip, wherein the housing comprises: a first sectioncomprising an outer face and an inner face, wherein the inner face ofthe first section comprises a channel to receive the test strip alongthe length of the first section, and a second section configured to beattached to the first section such that in an attached configuration theinner face of the first section and the test strip received in thechannel of the first section faces an inner face of the second section,wherein the second section comprises: a buffer port configured to allowa buffer solution to be dispensed to a portion of the test strip, a testwindow arranged such that one or both of a test location and a controllocation on the test strip are viewable through the test window in theattached configuration, a sample port disposed between the buffer portand the test window, and a set of projections disposed on the inner faceof the second section between the test window and the sample port suchthat in the attached configuration, each projection in the set ofprojections is in contact with the test strip, wherein a height of atleast one projection in the set of projections is different from aheight of another projection in the set of projections, and heights ofthe different projections are configured such that, in the attachedconfiguration, the set of projections produces a pressure gradientwithin the test strip that allows a fluid to flow at a predeterminedflow rate along the length of the test strip between the first and lastprojections in the set of projections; and a reader device configured toaccept at least a portion of the housing within the reader device,wherein the reader device comprises an optical system that captures oneor more images of one or both of the test location and the controllocation on the test strip, and in response, displays a result based onanalysis of the one or more images.
 2. The test system of claim 1,further comprising an identification tag associated with the housing,wherein the reader device is configured to query the identification tagto obtain at least a portion of identification information about apatient or sample associated with the test strip.
 3. The test system ofclaim 1, wherein the predetermined flow rate is such that the fluidflows from a portion of the test strip adjacent to the buffer port ofthe second section to a portion of the test strip adjacent to the testwindow of the second section in about 20 minutes.
 4. The test system ofclaim 1, wherein the set of projections is a set of ridges.
 5. The testsystem of claim 4, wherein the set of projections includes four ridges,a first ridge being disposed closer to the sample port than the otherridges, a fourth ridge being disposed closer to the test window than theother ridges, and a second ridge and a third ridge being disposedbetween the first and fourth ridges.
 6. The test system of claim 1,wherein distances between projections in the set of projections aresubstantially equal.
 7. The test system of claim 1, wherein the firstsection includes multiple attachment projections that are configured toattach to corresponding attachment receptacles disposed on the secondsection, wherein dimensions of the attachment projections and attachmentreceptacles are configured such that in the attached configuration, theset of projections produces the pressure gradient that allows the fluidto flow at the predetermined flow rate along the length of the teststrip.
 8. The test system of claim 1, wherein the test strip comprises:a base; a plurality of conjugates, each conjugate comprising a reportergroup bound to a first antibody that binds to ST2; a conjugate paddisposed along a length of the base, wherein the conjugate pad includesthe plurality of conjugates that bind with ST2 to produce conjugate-ST2complexes, and wherein the conjugate pad is configured to receive ablood plasma sample; a plurality of second antibodies that bind to ST2;a plurality of third antibodies that bind to the conjugate-ST2complexes; and a membrane disposed on the base such that the membrane isin fluid communication with the conjugate pad, wherein the plurality ofsecond antibodies are bound to the membrane in a test location and theplurality of third antibodies are bound to the membrane in a controllocation arranged further from the conjugate pad than the test location.9. The test system of claim 8, wherein either the first or the secondantibodies bind specifically to ST2.
 10. The test system of claim 8,wherein both the first and second antibodies bind specifically to ST2.11. The test system of claim 8, further comprising an absorbent paddisposed on the base and in fluid communication with the membrane at anend or side of the membrane opposite the conjugate pad, wherein theabsorbent pad is configured to absorb plasma and buffer that hastraversed through the membrane.
 12. The test system of claim 8, whereinthe conjugate pad is disposed on the base to receive a buffer solution.13. The test system of claim 8, wherein the conjugate pad comprisesfibers comprising glass, polyester, or both glass and polyester.
 14. Thetest system of claim 8, wherein at least one of the conjugate pad or themembrane comprises nitrocellulose.
 15. The test system of claim 8,wherein the first antibodies are monoclonal antibodies.
 16. The testsystem of claim 8, wherein the reporter group comprises fluorescentparticles.
 17. The test system of claim 8, wherein the third antibodiesare goat anti-mouse IgG antibodies.